This proposal focuses on Spt6 (168 kDa) and FACT, which is a heterodimer of Spt16 (119 kDa) and Pob3 (63 kDa). Spt6 and FACT are histone chaperones that are conserved throughout eukaryotes, implicated in HIV latency and other disease processes, and unlike most structurally characterized histone chaperones, are essential for viability. Both Spt6 and FACT function in the assembly of nucleosomes, which is required as the DNA is doubled during replication in S phase and also to replace nucleosomes that become irretrievably displaced by remodeling or by passage of an RNA polymerase during transcription. FACT also reorganizes nucleosomes, forming a loosened structure that promotes passage of RNA polymerase but maintains contacts with each of the components so that the same displaced histone molecules are incorporated back into the reconstructed nucleosome. Our collaboration has previously produced multiple structures of domains of Spt6, a complex of Spt6 with the essential factor Spn1, and multiple structures of domains of the FACT heterodimer. We also performed genetic and binding studies that validated the biological importance of these structures and advanced understanding of mechanisms. We will continue this multidisciplinary approach, which is essential for making meaningful progress in this challenging field. An important priority in Aims 1 and 2 is to biochemically and structurally characterize interactions with histones, which are fundamental to Spt6 and FACT function. We favor the model that Spt6 and FACT function in large part by covering histone surfaces that otherwise associate with DNA or other histones in the nucleosome structure, and we therefore believe that biochemically understanding these interactions and verifying their biological importance will reveal the primary basis of Spt6 and FACT activities in loosening and assembling nucleosomes. Histones are notoriously challenging subjects for binding studies, and our multidisciplinary approach, which includes rigorous biochemistry, places us in an excellent position to pursue high impact goals and has already led to an insightful preliminary FACT:H2A-H2B crystal structure. Specific hypotheses generated from the insights provided by Aims 1 and 2 are driving functional studies that are being pursued for Spt6 in Aim 3, including the mechanistic and physiological role of the Spn1 binding partner. Aim 4 is advancing the important but understudied question of how the essential histone chaperones are coupled to diverse biological pathways. Specifically, we have discovered that Spt6 binds directly and specifically with RNA polymerase II (RNAPII) and with Tom1. These interactions presumably underlie Spt6's distinct roles in transcription elongation and mRNA export, respectively, and the Tom1 interaction further implicates Spt6 in the maintenance of histone homeostasis and has ramifications for understanding certain forms of hepatocellular carcinoma and mental impairment in humans. Preliminary data include an initial EM structure of the RNAPII-Spt6 complex, identification of Spt6 point mutants that specifically disrupt binding to RNAPII or to Tom1, and identification of a phosphorylation- dependent switch for binding to RNAPII.